Method of selecting non-diabetogenic milk or milk products and milk or milk products so selected

ABSTRACT

The invention is based on the discovery that certain variants of β-casein may induce Type-1 diabetes in susceptible individuals while other variants do not. The invention consists of the selection of non-diabetogenic milk producing cows and recovering and processing their milk and milk products. Another aspect of the invention is selectively breeding cows which produce the non-diabetogenic milk.

This is a division of application Ser. No. 08/836,778 filed on Aug. 8,1997 now U.S. Pat. No. 6,451,368 issued Sept. 17, 2002, which is anational phase application filed under 35 U.S.C. §371 of InternationalApplication Number PCT/NZ95/00114 filed on Nov. 5, 1995, which claimspriority to New Zealand Application No. 264862 filed on Nov. 4, 1994.

TECHNICAL FIELD

This invention relates to a method for avoiding the triggering of Type 1diabetes in humans by the ingestion of milk or milk products. Moreparticularly, the method relates to the selection of milk which does notcontain a diabetogenic factor by selecting cows producing milk whichcontains any variant of β-casein which does not stimulate diabetogenicactivity in humans (a non-diabetogenic variant) to the exclusion of anyvariant of β-casein which does stimulate diabetogenic activity in humans(a diabetogenic variant).

BACKGROUND ART

Type 1 diabetes occurs in individuals who are genetically susceptible.However, even in identical twins, diabetes-may occur in one and not inthe other. The present invention relies upon the discovery of anenvironmental trigger for Type 1 diabetes which operates very early inlife.

The evidence that this environmental trigger is to be found in cows milkis based on epidemiological (Leslie et al, 1994), ecological (Virtanenet al, 1993) and animal experimental evidence (Elliott & Martin, 1984and Elliott 1992). The diabetogenic factor of the milk appears to be inthe casein fraction (Elliott et al, 1992), at least in the non-obesediabetic (NOD) mouse. Whey protein does not appear to, contain anydiabetogenic component (Elliott et al, 1992). It has been suggested thatbovine serum albumin (BSA), a protein found in the whey fraction of cowsmilk is the diabetogenic component of cows milk (Sheard, 1993). However,a review of the evidence supporting this theory does not indicate thatBSA was ever tested for diabetogenic activity in the absence ofβ-casein.

International PCT Application WO95/10537 discloses a method of producingdenatured bovine serum albumin milk products. It is stated that theconsumption of denatured BSA milk products tends to reduce thelikelihood of a person acquiring type 1 diabetes. However, there is noevidence presented of any trials where either human or animal subjectswere fed milk or milk products with denatured BSA. It relies upon thetheory mentioned above that BSA is the diabetogenic component of cowsmilk (Sheard, 1993). In European Patent Application 629,350 there isdescribed a method of hydrolysing cows milk protein to produce ahydrolysates substantially free of allergenic proteins. The hydrolysatealso is suggested to be useful in the prophylaxis and treatment of type1 diabetes melitis in children susceptible to such disease. In thedescription on page 6 of that specification it is suggested that BSA maybe a trigger to the immune system. However, there are no examples in thepatent specification and no reference to any papers showing any directevidence of this suggestion.

In South African patent specifications 61/1804 laid open on 28 Jun.1961, 61/2068 laid open on 20 Sep. 1961 and 62/600 laid open on 4 Jul.1962 there are described compositions alleged to be cures for diabetes.There are no examples, of any trials in support of these assertions. Thecompositions consist of casein as a base and fruit and leaves of SouthAfrica plants. It is inferable from the description that the activeingredient is the plant material and there is no mention that casein hasany role in causing or curing diabetes.

We have now tested the A1 and A2 variants of β-casein and a whey proteinon NOD mice and found that the A1 variant does have diabetogenicactivity while the A2 variant and whey protein do not show diabetogenicactivity.

It is an object of one aspect of the invention to use this finding to gosome way to selecting milk and milk products which do not contain adiabetogenic factor in such milk or milk product or at least to offerthe public a useful choice.

It is an object of another aspect of this invention to go some waytowards selectively breeding cows and bulls whose offspring produce milkwhich is not diabetogenic or which at least offers the public a usefulchoice.

DISCLOSURE OF THE INVENTION

Accordingly, the invention may be said broadly to consist in a method ofselecting milk for feeding to diabetes susceptible individuals whichcomprises testing milk from identified cows for the presence of variantsof β-casein and selecting those cows whose milk contains anynon-diabetogenic variant and does not contain any diabetogenic variant,and milking separately the non-diabetogenic variant milk producing cowsand recovering and maintaining their milk separately from milk from anyother source.

Preferably said non-diabetogenic variant is the A2 variant of β-casein.

Alternatively said non-diabetogenic variant is the A3, D or E variant ofβ-casein.

Preferably said diabetogenic variant is the A1 variant of β-casein.

Alternatively, said diabetogenic variant is any one of the B, C and Fvariants.

Preferably, said recovered milk is tested for the presence of anydiabetogenic variant and discarded if any is found.

Alternatively, said method of testing, comprises the use of massspectrometry.

In one embodiment said mass spectrometry comprises electro sprayeionisation mass spectrometry.

Alternatively, said mass spectrometry comprises fast-atomic bombardmentmass spectrometry.

Preferably, said method of testing comprises polyacrylamide gelelectrophoresis using an acid urea gel.

Preferably, said process includes the additional step of processing saidmilk into milk products.

There are a large number of processes known to those skilled in the artfor converting milk into milk products. These range from separatingcream from whole milk to produce skim milk through to the use ofmicrofiltration and ultrafiltration to produce a wide range of productssuch as those described in international application PCT/NZ95/00086, thespecification, claims and drawings of which are incorporated herewith byreference.

One particular product of interest from the aforementioned internationalapplication is milk protein concentrate. This may be prepared by otherprocesses such as that described in IDF Special Issue No. 9201, (1991),Chapter 5 entitled “Milk Protein Concentrate”, A. Novak.

Another milk product according to the invention is casein derived fromnon-diabetogenic milk by any well known casein producing process such asdescribed in Southward et al, 1980.

The invention may be said broadly to consist in milk selected accordingto the process herein above defined.

The invention may also be said broadly to consist in a non-diabetogenicmilk product prepared by any one of the processes described hereinabove.

The invention may also be said broadly to consist in a method forreducing the risk of contracting type 1 diabetes in a susceptibleindividual which comprises restricting the milk or milk product intakeof that individual to milk containing only a non-diabetic variant ofbeta casein.

Preferably, said susceptible individual is an infant or young child.

The invention may also be said broadly to consist in a method ofselecting milk for feeding to a Type-1 diabetes susceptible individualwhich comprises testing milk from identified cows for the presence ofthe hexapeptide Pro-Gly-Pro-Ile-His-Asn (SEQ ID NO: 1), or a proteinfragment containing the hexapeptide Pro-Gly-Pro-Ile-His-Asn (SEQ IDNO: 1) and selecting those cows whose mild does not contain saidhexapeptide or said protein fragment containing said hexapeptide, andmilking separately the cow whose milk does not contain the saidhexapeptide or said protein fragment containing said hexapeptide andmaintaining their milks separately from milk from any other source.

Preferably, said separated milk is also tested for the presence of saidhexapeptide or said protein fragment containing said hexapeptide and anymilk which does contain said hexapeptide or said protein containinghexapeptide is discarded.

Preferably, the method of testing for said hexapeptide is by usingchromatographic purification of said hexapeptide followed by amino acidsequencing.

Preferably, said process includes the additional step of processing saidmilk into milk products.

The invention may be said broadly to consist in milk selected accordingto the process herein above defined.

The invention may also be said broadly to consist in a non-diabetogenicmilk product prepared by any one of the processes described hereinabove.

Preferably, said susceptible individual is an infant or young child.

In another embodiment the invention may be said broadly to consist in amethod for selecting breeding cows which produce daughters whose milk isnot diabetogenic to susceptible children which comprises determining thegenotype of said cows and selecting those whose daughters produce milkwhich does not contain the diabetogenic factor present in β-casein.

Alternatively, the invention may be said broadly to consist in a processfor selectively breeding bulls which produce daughters whose milk doesnot contain the diabetogenic factor present in β-casein which comprisesdetermining the genotype of said bulls and selecting those whichdaughters which produce milk which does not contain the diabetogenicfactor present in β-casein.

Preferably, the phenotyping of daughters to determine the genotype ofsaid bull is done by testing the milk of said daughters for absence ofdiabetogenic variants of β-casein and the presence of non-diabetogenicvariants of β-casein.

Alternatively, said cows or bulls are genotyped directly by usingappropriate probes and polymerase chain reaction technology.

In another embodiment the invention may be said broadly to consist incows selected in accordance with the immediately preceding method.

In a still further embodiment the invention may be said broadly toconsist in bulls selected in accordance with the above defined method.

In a still further embodiment the invention may be said broadly toconsist in semen of bulls selected in accordance with the above definedmethod.

In an alternative to any of the above processes or products the milk ormilk product is goat's milk or milk product, sheep's milk or milkproduct, buffalo's milk or milk product, or milk or milk product fromany other mammal which is fit for human consumption.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a photograph of PAGE bands of β-casein variants.

FIG. 2 is a protein sequence representation of the bovine β-casein A1variant (SEQ ID NO: 2).

FIG. 3 is a bar graph of differences in antibody levels to mixedβ-casein variants in human patients of different ages with recentlydiagnosed diabetes against controls.

MODES OF CARRYING OUT THE INVENTION

Milk Protein Polymorphism

Following the initial discovery by Aschaffenburg and Drewry (1955) thatthe major whey protein in milk, β-lactoglobulin is found in a number ofvariant forms, all major milk proteins (α_(S1)-casein, α_(S2)-casein,β-casein, κ-casein and α-lactalbumin) are now also known to exist as anumber of variant protein species, due to genetic polymorphism at thegene loci coding for these proteins (Grosclaude, 1988, Ng-Kwai-Hang andGrosclaude, 1992). The primary sequences of the casein proteins and acomprehensive list of the amino acid sequence changes that give rise tothe variant forms of these proteins is given by Swaisgood (1992),Ng-Kwai-Hang and Grosclaude (1992) and Visser et al (1995). The proteinβ-casein has eight variant forms:

-   -   A1 A2 A3 B C D E F

As highlighted by Ng-Kwai-Hang and Grosclaude (1992) the A2 variant isconsidered to be the original variant type of the Genus Bos.

Because the milk protein genes are expressed codominantly (Mepham et al,1992), individual cows produce milk containing either a single variantform of β-casein (A1A1, A2A2, A3A3 phenotype cows etc) or a mixture oftwo variant forms of β-casein (A1A2, A1A3, A2A3 phenotype cows etc). Thefrequency of β-casein phenotypes in a sample population of New Zealanddairy cows expressed as percentages is set out in Table 1.

TABLE 1 Phenotype A1- A1- A1- A2- A2- Breed A1 A2 A3 A1B A2 A3 A2B BBFriesian 21 43 0.1 5 24 0.16 5.6 0.4 Jersey 0 9 0 5 46 0 34 6

The predominant β-casein variant from the Indian cow (Bos indicus) isidentical in its amino acid sequence to the A2 variant of the commondairy cow (Bos taurus). The Maasai people of Central Africa drink largequantities of cows milk from Bos indicus from very early life, but donot develop diabetes, whereas Finnish people who drink similarly largequantities of milk from mixed Bos taurus herds have a very high diabetesincidence.

Although the examples of this specification relate to milk from cowsthere is no reason to doubt that the processes and products describedare equally applicable to milk from any mammalian source which is fitfor human consumption.

EXAMPLE 1 Finnish and Samoan Diets Fed to NOD Mice

Based on these observations we have assessed the effect of human infantdiets given to 1 year old normal Finnish children (who have a subsequentrisk rate of diabetes of 40/100,000/yr in the following 15 years), andsimilar diets collected from Samoan children (in which the subsequentexpected diabetes incidence is <1/100,000/yr) on the diabetes prone NODmouse.

Diets from Finnish and W. Samoan children were collected from 10children from each location. The children were 10–14 months old. Thefamilies were cooperative middle-class and the collections weresupervised by a trained dietitian, who also asked the parents to providea record of the types and quantities of food given.

As each portion of food or drink was given to the infant a similarquantity was placed in a container. This was kept frozen until a totalof 10 days equivalent food was collected. Thereafter the food was freezedried and then shipped to New Zealand. The food was sterilized by gammaradiation before being given to the mice.

Children in these two groups consumed <100 ml and >500 ml of cows milkper day respectively in Samoa and Finland.

The diets were then fed to groups of NOD mice from weaning either as thesole dietary or as a 10% w/w addition to a soy based infant formulaProsobee™ previously shown not to cause diabetes in this strain ofmouse. The diabetes incidence in the succeeding 250 days was thenassessed. The results are shown in Table 2. The Finnish diet is more(about threefold) diabetogenic than the Samoan diet and this correspondsapproximately to the relative proportion of cows milk in the two diets.

TABLE 2 Effect of duplicate infant diets from Finland and Western Samoaon diabetes incidence in NOD mice fed these diets from weaning. No. ofAnimals Developing “p“ Value No. of Diabetes by (Fisher Exact DietAnimals 250 days % Test) 1 a) Western Samoan diet 24 14 58 avb b)Finnish diet 20 16 80 0.112 2 a) 10% Western Samoan 43 4 9 avb diet inProsobee ™ IF .0007* b) 10% Finnish diet in 39 13 33 Prosobee ™ IF 3Prosobee ™ IF alone 33 0 0 Prosobee ™ IF v2a.113 Prosobee ™ IF v2b.00Q*

The “p” value is the statistical estimate of the strength of theobservation that there is a difference between the two groups. Ingeneral a “p” value of less than 0.05 is accepted as highly significant,and has been asterisked. The other symbols in each box refer to thecomparison being made e.g. in the first box “a” refers to 1a i.e. theproportion 14/24 and the symbol “b” to the proportion 16/20 the “v” ismerely “versus”. In this instance the p value of 0.112 means that thisresult could have arisen by chance 11.2 times out of 100. IF meansinfant formula.

EXAMPLE 2 NOD Mice Fed Hydrolysed Casein and Casein

Similar groups of mice were fed a diet whose sole nitrogen source washydrolysed casein or casein itself. Only the casein fed mice developeddiabetes (Table 3).

TABLE 3 Effect of casein hydrolysed infant formula (Pregestimil ™) andsimilar intact casein infant formula (Portagen ™) on diabetes incidencein NOD mice fed these diets from weaning No. of Animals “P” Value No. ofDeveloping (Fisher Exact Type Animals Diabetes Percentage Test)Hydrolysed 49 1 2 .001* casein Intact casein 29 8 28

EXAMPLE 3 NOD Mice Fed A₁ and A₂ Variants of Cow β-Casein and WheyProteins

Similar groups of mice were fed a diet of the soy infant formulaProsobee™ and 10% W/W of:

a) β-casein A1 variant,

b) β-casein A2 variant,

c) whey,

d) Bos indicus casein, or

e) a 50/50 mixture of β-casein A1 variant/A2 variant.

The subsequent diabetes incidence in the groups is shown in Table 4. Theβ-casein A1 variant precipitated diabetes in 9 or 20 cases, the A2variants in no cases and the A1/A2 mixture in 4 of 20 cases. The resultmid-way between the A1 and A1 alone results is consistent with therebeing no interaction between A1 and A2. Only seven percent of the wheyprotein fed mice developed diabetes.

TABLE 4 Effect of bovine β-casein A1 and A2 variants and whey proteinson diabetes incidencein NOD mice fed these proteins as 10% additions toProsobee ™ infant formula from weaning Number of Animals NumberDeveloping “P” Value of Diabetes by 250 (Fisher Exact Animals daysPercentage Test) a) 10% β-casein 20 9 45 avb .001* A1 variant avc .003*b) 10% β-casein 18 0 0 bvc N.S. A2 variant c) 10% whey 29 2 7 d) 10% Bos20 0 0 bvd N.S indicus casein* e) 10% β-casein 20 4 20 A1/A2 variants(equal mixture) *Known to contain only the A2 variant (see Example 7)

From the animal experiments in Examples 1 to 3 it can be concluded thatmixed dairy casein is diabetogenic in the NOD mouse and that thisproperty resides in the β-casein A1 variant.

EXAMPLE 4 Acid Urea Method for Typing Milk for β-Casein Variant

The polyacrylamide gel electrophoresis (PAGE) was carried out on aBiorad Mini Protean II system (supplied by Biorad Laboratories,Hercules, Calif., USA). The method separates qualitatively variants ofβ-casein by net charge and molecular weight.

All the reagents are analytical grade unless otherwise stated.

28.5 ml of glacial acetic acid was diluted to 500 ml with deionisedwater (Milli-Q water) to make the buffer. 500 ml of buffer was used perrun.

Acid Urea Stacking Gel Solution

The acid urea stacking gel solution was made up by weighing up thefollowing reagents and dissolving them in Milli-Q water to approximately80 ml. The pH was adjusted to 4.1 with acetic acid and made up to 100 mlin a measuring cylinder.

-   36.04 g urea-   6.0 g acrylamide/bis premix (5% C) Serva-   0.113 g ammonium acetate-   0.178 g thiourea-   1.48 ml glacial acetic acid

Acid Urea Resolving Gel Solution

The following reagents were dissolved in Milli-Q water to approximately130 ml, the pH adjusted to 3.86 with acetic acid and made up to 150 mlin a measuring cylinder.

-   9.75 g Acrylamide/bis premix (5% C) Serva Research Grade-   40.36 g urea-   9.48 ml glacial acetic acid-   0.72 g ammonium acetate-   0.26 g thiourea

Acid Urea Sample Buffer

An acid urea sample buffer was made up by weighing out the followingreagents and dissolving them in approximately 350 ml of Milli-Q water.The pH was adjusted to 4.16 with acetic acid and the volume made up to400 ml.

-   163.18 g urea-   6.69 ml glacial acetic acid-   0.451 g ammonium acetate

A solution of Bromophenol Blue (0.4 w/v) was made up by dissolving 1.6gins of bromophenol blue with 6.8 ml of NaOH (0.1 m). This was then madeup to 400 ml with Milli-Q water.

A distaining solution was made up by mixing 8 liters of Milli-Q water,1000 ml of glacial acetic acid and 1000 ml of isopropanol.

A Coomassie blue R stain was made up by dissolving 1.00 gms of brilliantblue R in 500 ml isopropanol adding 200 ml of glacial acetic acid andmaking up to 2 liters with Milli-Q water. The solution was covered witha gas tight cling wrap and stirred overnight. The stain was filteredusing a Buchner funnel and Whatman number 1 filter paper.

The resolving gel solution was placed between the glass plates of anelectrophoresis apparatus. The stacking gel was also placed between theglass plates and allowed to polymerise according to known techniques.

The β-casein variant samples were added in 25 μl aliquots to 750 μl ofsample buffer. 10 μl of 2-mercaptoethanol was added and the samplesallowed to stand at least one hour at room temperature or 4° C.overnight. The samples were run in sample slots. The samples wereinjected by syringe.

The power pack of the apparatus was set to deliver:

Program = T/V-H Current = 70 mA Field strength = 210 V Watt power = 6.5Time = 1 hour for 2 gels

After the running time of one hour the power pack was turned off and thepower cables disconnected. The gel was pealed off the plate of theapparatus into the container of stain (approximately one hour). Afterdistaining in distaining solution (less than one hour) the gel containerwas put onto a light box and a photograph taken to identify the bands.

The bands as photographed are shown in FIG. 1. The samples in the lanesare identified. The banding pattern for β-casein variant proteins on anacid urea PAGE gel is, starting from the top, A3 variant, A2 variant, A1variant and B variant.

EXAMPLE 5 Reverse Phase HPLC and Mass Spectromatic Analysis Method ofTyping Milk for β-Casein Variant

β-casein variants in milk may be typed using the method of Visser et al(1995). In Visser's paper the F variant was identified by using reversedphase HPLC followed by electrospray ionisation and fast atom bombardmentto determine molecular weights and characterize the amino acidsubstitutions in the protein. The other variants can be determined usingthe same techniques.

EXAMPLE 6 Direct Genotyping of Cows or Bulls

DNA extracted from frozen bull semen was directly sequenced bypolymerise chain reaction (PCR) to detect the sequences for bovineβ-casein variants according to the method of Lien et al (1992).

Semen containing the non-diabetogenic alleles detected can be used in abreeding programme.

EXAMPLE 7 Separation of β-Casein A1 and A2 Variants

1. Phenotype Identification

Cows (Bos taurus) homozygous for the β-casein variant A1 and A2 genes(β-casein A1A1 and A2A2 phenotype cows) were identified bypolyacrylamide gel electrophoresis (PAGE) of milk samples fromindividual cows using the acid urea gel system as described in Example4.

Samples of New Zealand casein (Alacid 710™) and casein prepared from themilk supplied from a herd of Australian Bos indicus cows were alsosubjected to PAGE as described above in Example 4. Alacid 710™ casein ismanufactured from bulk New Zealand milk, which in turn is produced frommany thousands of cows of different β-casein phenotypes.

The relative amounts of the different genetic variants of β-casein inAlacid 710™ casein were determined by computing densitometry in a methoddescribed by Hill (1993) and Singh and Creamer (1991). The results aretabulated in Table 5.

TABLE 5 β-casein Variant % Amount in Alacid 710 Range (N = 9) A1 40.038.4–41.4 A2 51.5 48.1–53.3 B 7.5 6.5–8.5 A3 1.0 0.2–1.7The Australian Bos indicus casein was found to contain only the β-caseinA2 variant.

2. Milk Production, Segregation and Collection

From a total of 3183 cows located on 25 large farms in the Manawatu andWaikato regions of New Zealand, approximately 400 cows were selected andplaced on a single farm as a mixed herd such that the β-casein A1A1 andA2A2 phenotype cows in this herd were subjected to identical farmmanagement and feeding practices. Milk supplied from either β-caseinA1A1 or A2A2 phenotype cows was segregated by the use of dual milk linessituated in the farm milking parlour and collected in separaterefrigerated vats essentially as described by Hill (1993). The bulkedβ-casein A1A1 and A2A2 phenotype milks were then pumped into separatecompartments of milk tanker before tankering to the New Zealand DairyResearch Institute Pilot Plant.

3. Casein Manufacture

Lactic casein was manufactured in the New Zealand Dairy ResearchInstitute Pilot Plant from the β-casein A1A1 and A2A2 phenotype bulkmilks using methods essentially identical to those used in a standardNew Zealand commercial lactic casein manufacturing dairy factory (seeMuller, 1986). These caseins together with other constituents were thenused in the feeding trials as described in Examples 2 and 3.

EXAMPLE 8 Immunostimulating Peptides from β-Casein

Partially hydrolysed mixed caseins containing a variety of peptides arealso diabetogenic, whether given in soy formula in the diet or byinjection into mice fed on the soy formula. This suggests that theintact casein molecules may not be necessary for the diabetogenicaction. Among the peptides resistant to hydrolysis is the hexapeptidePro-Gly-Pro-Ile-His-Asn (SEQ ID NO: 1) shown in FIG. 2.

Peptides of β-casein have been found to stimulate the human immunesystems (Meisel and Schlimme, 1990; Fox and Flynn, 1992). M'HamielJzairi et al (1992) have shown that there is a specific binding site onthe human macrophage for the immunostimulating peptide Val-54 to Tyr-59from human β-casein. Although the hexapeptide Pro-63 to Asn68 frombovine β-casein (highlighted in the complete β-casein A1 variant aminoacid sequence shown in FIG. 2) has been found to stimulate thephagocytic activity of murine macrophages (Migliore-Samour et al, 1989)the binding of this peptide to human macrophages has until now not beendemonstrated.

Using the method described by M'Hamiel Jaziri et al (1992) the humanmacrophage binding of the bovine peptides Pro-63 to Asn-68 (from theβ-casein A1 and A2 variant sequences) were studied and compared with thebinding of the peptide Val-54 Tyr-59 from human β-peptide to each of anormal human macrophage and a prediabetic human macrophage. The resultsare set out below in Table 6. They show that both bovine A1 and A2 andhuman hexapeptides bind specifically both to normal human macrophagesand to prediabetic macrophages, implying some immune function.

With the small numbers involved (three subjects in each group) the“prediabetic” macrophages bind the peptides with an avidity order ofA1>A2>human, whereas the normal macrophages bind A1=A2=human. A2 isbound to the same extent by normal and prediabetic macrophages. Thismeans that the prediabetic macrophages are more likely than normals topresent the A1 peptide to the immune system, which may account for thehigher levels of antibody to the A1 casein variant found in newlydiagnosed diabetics.

TABLE 6 Binding of tritiated hexapeptides to PB monocyte/Macrophages(+Glucose, NaN3, Captopril, Benzylsuccinate, Elastatinal) Peptide CPMRatio */** NORMAL PBM (N = 3) Human 2750* 1.2 Bovine A1 2220** A2 2900PREDIABETIC PBM (N = 3) Human 1400* 0.31 Bovine A1 4500** A2 2750

The β-casein A3, D and E variants each contain an identical hexapeptidesequence between Pro-63 and Asn-68 to that found in the β-casein A2variant (proline at position 67), however the β-casein A1, B, C and Fvariants proline-67 is substituted by a histidine residue.

Antibodies to mixed caseins are found at higher levels in newlydiagnosed diabetics (IDDM) than in normal controls (FIG. 3) and thisdifference appears to reside in the higher levels of antibodies directedagainst A1 rather than A2 caseins in the diabetics. Testing of theantibodies was done by Enzyme Immuno-Assay. In this the caseins arebound to the wall of a plastic container and the serum containing theantibodies then brought into contact with this bound casein. Afterincubation the serum is decanted and any antibody present will havebecome bound to the casein. This bound antibody is then measured by acolour reaction.

From the above it can be concluded that cows milk β-casein A1 variant isdiabetogenic in the NOD mouse whereas the β-casein A2 variant is not. Adigestion resistant fragment of these caseins can bind to antigenpresenting cells in humans and in the case of diabetics an increasedresponse to the β-casein A1 variant can be observed.

Together with the epidemiological observations on the difference indiabetes incidence in children receiving large amounts of β-casein A1and A2 variants such as found in the Finish diet compared with thosereceiving only a β-casein identical with A2 variant, (the Maasai), it islikely that β-casein A1 variant is the principal moiety in cows milkwhich is diabetogenic in humans.

EXAMPLE 9 The Measurement of Anti-Casein Antibodies in Newly DiagnosedDiabetics and Age Matched Normals

Antibodies were measured by an ELISA technique utilising immunoglobulinG subclass measurement to develop the class specific antibody levels.

TABLE 7 Levels of Antibodies to Purified A1 and A2 Beta CaseinClassified by Subtype of IgG Diabetics are at onset (children normals(age age 1–12 yr) matched) Diabetic Normal IgG class (OD) 1 2 3 4 1 2 34 #1 A1 .086 0 0 0 0 0 0 0 A2 .072 0 0 0 0 0 0 0 #2 A1 .029 0 .038 0 0 00 0 A2 0 0 .022 0 0 0 0 0 #3 A1 .106 .048 .138 .610 0 0 0 0 A2 .045 .056.104 .529 0 0 0 0 #4 A1 0 0 0 0 0 0 0 0 A2 0 0 0 0 0 0 0 0 #5 A1 .021 0.237 0 0 0 0 0 A2 0 0 .250 0 0 0 0 0 #6 A1 .098 .042 .025 1.374 0 0 0 0A2 .052 .022 0 1.110 0 0 0 0 #7 A1 .075 0 0 .165 0 0 0 .083 A2 .057 0 0.099 0 0 0 .052 #8 A1 .128 0 .028 .219 0 0 0 0 A2 .068 0 0 .066 0 0 0 0#9 A1 .210 .049 .195 .397 0 0 0 .03 A2 .197 .043 .155 .397 0 0 0 0 #10A1 .669 0 .286 .539 0 0 0 0 A2 .606 0 .272 .525 0 0 0 0 #11 A1 0 0 .171.162 .249 .041 .213 .052 A2 0 0 .144 .171 .272 .043 .174 .044 #12 A1.045 0 .061 .039 .372 0 .385 .233 A2 0 0 .046 0 .256 0 .362 .031 #13 A1.023 0 .066 0 .051 0 .026 0 A2 0 0 .046 0 .04 0 .026 0 #14 A1 0 0 0 0 00 .056 .021 A2 0 0 0 0 0 0 .051 0 #15 A1 .042 0 .124 .035 .083 0 .242.129 A2 .054 0 .114 .036 .071 0 .246 .129 #16 A1 .154 0 .234 .061 0 0.092 0 A2 .106 0 .234 .051 0 0 .103 0 #17 A1 .132 .069 .285 .139 .073 0.147 .079 A2 .097 .052 .254 .153 .063 0 .109 .025 #18 A1 .076 0 .131.059 .075 0 .116 .029 A2 .062 0 .108 .049 .069 0 .109 .025 #19 A1 .049 0.056 .023 .044 .056 .056 .097 A2 .051 0 .050 .020 .034 .046 .052 .091#20 A1 .149 0 .220 .077 .237 .304 .333 .294 A2 .106 0 .188 .056 .227.277 .325 .273 Best discriminant is IgG1 + 3 against A1 beta casein18/20 v 9/20 (p = .003) IgG2 + 4 13/20 v 9/20 (p = .204)

The ratio of response of IgG1+3 to the two beta caseins also appears tobe different between diabetics and normals with those of the diabeticsbeing higher.

(9/20 diabetics have a ratio of >1.3 whereas 0/20 have this high a ratioamong the normals (p=0.001)

IgG (1+3) represents a Th1 (‘helper’) response whereas (IgG+4)represents a Th2 (‘suppressor’) response.

From this data it appears likely that diabetics more often than normals:

1) mount an immune response to beta caseins,

2) that these are more often an IgG1+3 response than a IgG2+4 response,and

3) that these responses are more often directed to A1 beta casein thanthe A2.

REFERENCES

-   Aschaffenburg R and Drewry J (1955) Nature, 176, 218–219-   Ng-Kwai-Hang, K F, Hayes, J F, Moxely, J E and Monardes, H G (1984)    J Dairy Sci, 67 835–840-   Creamer, L K (1991) Bulletin of the International Dairy Federation    Advanced Dairy Chemistry—Vol 1: Proteins, 261. International Dairy    Federation, Brussels, Belgium-   Grosclaude F (1988) Productions Animates, INRA 1, 5–17-   Fox P F and Flynn A (1992) Advanced Dairy Chemistry—Vol 1: Proteins    (Ed. Fox P F) Elsevier Science Publishers Ltd, London, pp 255–284-   Hill J P (1993) J Dairy Sci, 76 281–286-   Meisel H and Schlimme E (1990) Trends in Food Science and    Technology, X, 41–43-   Mepham T B, Gaye P, Martin, P and Mercier J C (1992) Advanced Dairy    Chemistry—Vol 1: Proteins (Ed. Fox P F) Elsevier Science Publishers    Ltd, London, pp 491–543-   M'hamiel Jaziri et al (1992) Biochim, Biophys, Acta, 1160 251–261-   Migliore-Samour D, Floch and Jolles P (1989) J Dairy Res, 56,    357–362-   Muller L L (1986) Developments in Dairy Chemistry—1: Proteins (Ed.    Fox P F) Elsevier Science Publishers Ltd, London, pp 315–337-   Ng-Kwai-Hang, K F and Grosclaude F (1992) Advanced Dairy    Chemistry—Vol 1: Proteins (Ed. Fox P F) Elsevier Science Publishers    Ltd, London, pp 405–455-   Singh H and Creamer L K (1991) J Dairy Res, 58, 269-   Swaisgood H E (1992) Advanced Dairy Chemistry—Vol 1: Proteins (Ed    Fox P F) Elsevier Science Publishers Ltd, London, pp 63–110-   Sheard N F (1993) Nutrition Reviews, 51 79–89-   Leslie, R D & Elliott, R B (1994): Early Environmental Events As a    Cause of IDDM—Evidence & Implications. Diabetes 43, 843–850.-   Virtanen, S M; Rasanen L; Ylonen K; Aro A; Clayton D; Langholz B;    Pitkaniemi J; Savilahti E; Lounamaa R; Tuomilehto J; Akerblom, H K;    Childhood Diabetes in Finland (1993): Early Introduction of Dairy    Products Associated with Increased Risk of IDDM in Finnish Children.    The Childhood in Diabetes in Finland Study Group. Diabetes 42 (12,    Dec), 1786–1790.-   Elliott R B & Martin J M (1984): Dietary Protein: A Trigger of    insulin-dependent diabetes in the B B Rat? Diabetologia 26, 297–299.-   Elliott R B (1992): Epidemiology of Diabetes in Polynesia and New    Zealand. In: Epidemiology and etiology of Insulin-Dependent Diabetes    in the Young. Vol 21. (Eds: Levy-Marchal, C; Czernichow, P),    Pediatr. Adosesc. Endocrinol, Karger, Basal, 66–71-   Elliott R B, Bibby N; Reddy S (1992): “Casein Peptide Precipitates    diabetes in the NOD Mouse and Possibly Humans”. In Genetic and    Environmental Risk Factors for Type 1 Diabetes (IDDM), Including a    Discussion on the Autoimmune Basis. Eds: Laron Z and Karp M Freund    Publishing House Ltd.-   S Lien, P Alestrom, H Klungland and S Rogne (1992) Animal Genetics    23, 333–338.-   Southward, C R & Walker N L (1980) The Manufacture and Industrial    Use of Casein. New Zealand Journal of Dairy Science and Technology,    15, 201–217.-   Servaas Visser, Charles J Slangen, Fija M Lagserwerf, William D Van    Dongen, Johan Haverkamp: Identification of a new genetic variant of    bovine β-casein using reversed-phase high-performance liquid    chromatography and mass spectrometric analysis, Journal of    Chromatography A, 711 (1995)141–150.

1. A method for selecting cows whose milk is not diabetogenic tosusceptible individuals which comprises determining the genotype of saidcows and selecting those which produce non-diabetogenic milk whichcontains a β-casein variant with proline at amino acid position 67 anddoes not contain diabetogenic varients of β-casein which have ahistidine at amino acid position
 67. 2. A method for selecting bullswhich produce daughters whose milk contains a β-casein variant withproline at amino acid position 67 which comprises determining thegenotype of said bulls and selecting those whose daughters produce milkwhich contains a β-casein variant with proline at the 67 position. 3.The method as claimed in claim 1 wherein said cows are genotypeddirectly by using an appropriate probe and a polymerase chain reaction.4. The method as claimed in claim 2 wherein semen from a bull beingtested is genotyped directly by using an appropriate probe and apolymerase chain reaction.
 5. The method as claimed in claim 1 whereinsaid β-casein variant is the A2 variant of non-diabetogenic β-casein. 6.The method as claimed in claim 2 wherein said β-casein variant is the A2variant of β-casein.
 7. The method as claimed in claim 1 wherein saidβ-casein variant is selected from the group consisting of the A3, D andE variants of non-diabetogenic β-casein.
 8. The method as claimed inclaim 2 wherein said β-casein variant is selected from the groupconsisting of the A3, D and B variants of β-casein.